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Abstract

Introduction

We investigated the clinical and serological features of patients with antineutrophil
cytoplasmic antibody (ANCA)-associated vasculitis (AAV) in Japan using data from a
nationwide, prospective, inception cohort study.

Methods

In total, 156 Japanese patients with newly diagnosed AAV were classified according
to the European Medicines Agency (EMEA) algorithm with exploratory surrogate markers
for AAV-related non-granulomatous pulmonary lesions, predefined as alveolar haemorrhage
and interstitial lung disease (ILD), and their clinical and serological features were
evaluated.

Results

Using the EMEA algorithm, we identified 14 patients (9.0%) with eosinophilic granulomatosis
with polyangiitis (EGPA), 33 (21.2%) with granulomatosis with polyangiitis (GPA),
78 (50.0%) with microscopic polyangiitis and renal-limited vasculitis (MPA/RLV), and
31 (19.9%) with unclassifiable vasculitis. The average ages of patients with EGPA
(male/female, 5/9), GPA (12/21), and MPA/RLV (35/43) and unclassifiable (9/22) were
58.0, 63.6, 71.1, and 70.6 years, respectively. Myeloperoxidase (MPO)-ANCA and proteinase-3
ANCA positivity was 50.0% and 0% for EGPA, 54.6% and 45.5% for GPA, 97.4% and 2.6%
for MPA/RLV, and 93.5% and 3.2% for unclassifiable, respectively. According to the
Birmingham Vasculitis Activity Score (BVAS), cutaneous (71.4%) and nervous system
(92.9%) manifestations were prominent in EGPA and ear, nose, and throat manifestations
(84.9%) and chest manifestations (66.7%) in GPA. Renal manifestations developed frequently
in MPA/RLV (91.0%) and GPA (63.6%). The average serum creatinine levels were 0.71 mg/dL
for EGPA, 1.51 mg/dL for GPA, 2.46 mg/dL for MPA/RLV, and 0.69 mg/dL for unclassifiable.
The percentages of patients with ILD were 14.3% for EGPA, 9.0% for GPA, 47.4% for
MPA/RLV, and 61.3% for unclassifiable. Patients with ILD (n = 61) had significantly lower BVAS (P = 0.019) with fewer ear, nose, and throat and cardiovascular manifestations than
patients without ILD (n = 95).

Conclusions

MPO-ANCA-positive MPA/RLV is the most common form of AAV in Japanese patients, and
one-half of patients with GPA were positive for MPO-ANCA. ILD is an important clinical
manifestation in Japanese patients with AAV. Unclassifiable vasculitis with MPO-ANCA
positivity and ILD may represent a novel variant of MPA.

Trial Registration

Introduction

Microscopic polyangiitis (MPA), granulomatosis with polyangiitis (Wegener’s granulomatosis)
(GPA), and eosinophilic granulomatosis with polyangiitis (Churg–Strauss syndrome)
(EGPA) are the major categories of antineutrophil cytoplasmic antibody (ANCA)-associated
vasculitis (AAV), a multisystem autoimmune disease characterised by ANCA production
and small-vessel inflammation [1,2]. Despite the overlapping clinicopathologic characteristics between the component
diseases, the disease evolution, organ involvement, prognosis, and other clinical
characteristics differ substantially among them. In addition, there are interesting
geographic and ethnic differences in their relative incidence and myeloperoxidase
(MPO)-ANCA or proteinase-3 (PR3)-ANCA positivity [3].

In 2007 Watts and colleagues proposed an AAV classification algorithm, the European
Medicines Agency (EMEA) algorithm, with consensus of a group of European physicians
interested in the epidemiology of vasculitis [4]. This stepwise algorithm incorporated both the American College of Rheumatology (ACR)
criteria for EGPA and GPA and the Chapel Hill Consensus Conference (CHCC) definition
of EGPA, GPA, and MPA [2]. In the EMEA algorithm, surrogate markers of granulomatous inflammation for GPA and
those of renal vasculitis for renal-limited vasculitis (RLV), an organ-limited variant
of MPA, were defined [4]. This algorithm is useful for classifying patients with AAV because no overlapping
diagnoses occur and fewer patients are considered to have unclassifiable vasculitis
[5], and has been used as the standard method for classification of AAV diseases in recent
studies [6,7].

Only two reports have validated the algorithm in other ethnicities outside Europe
using a good-quality database. Studies from China [5] and Japan [3] applied the EMEA algorithm to their patient populations and found that MPO-ANCA-positive
MPA was the most common form of AAV. These studies, however, were retrospective and
evaluated clinical data of patients from a small number of hospitals.

The lung is one of the organs frequently involved in AAV, and pulmonary granuloma,
alveolar haemorrhage, and interstitial lung disease (ILD) are representative pulmonary
lesions. Among these, only pulmonary granuloma is included in the EMEA algorithm.
ILD in AAV is associated with MPO-ANCA and is more common in Asian countries [8-10] than in western countries [11,12], and some patients with MPO-ANCA and ILD subsequently develop typical MPA [13]. To understand the nature of AAV and classify the disease from a global perspective,
it is essential to more precisely delineate the clinical implications of ILD in AAV
in Asian countries.

To characterise the clinical and laboratory features, effectiveness, and safety of
the remission-induction therapy used, as well as the prognosis of Japanese patients
with AAV, the Research Committee on Intractable Vasculitides of the Ministry of Health,
Labour and Welfare of Japan implemented a nationwide prospective cohort study of Remission
Induction Therapy in Japanese Patients with ANCA-associated Vasculitides (RemIT-JAV).
In this study, we classified Japanese patients with newly diagnosed AAV enrolled in
the RemIT-JAV study according to the EMEA algorithm and compared their phenotypes
across the AAVs. We also investigated the clinical relevance of ILD in the patient
population.

Methods

Database

Twenty-two tertiary care institutions (university hospitals and referring hospitals)
participated in this study (See Appendix) and enrolled consecutive patients with newly
diagnosed AAV from April 2009 to December 2010. The criteria for enrolment in this
study included receiving a diagnosis of AAV from the site investigators, fulfilling
the criteria for primary systemic vasculitis proposed by the EMEA algorithm [4], and requiring immunosuppressive treatment based on the discretion of the site investigators.
The exclusion criteria were age younger than 20 years, recurrent AAV, serological
evidence for hepatitis B virus or hepatitis C virus infection, and a history of malignancies
because this may influence treatment selection and prognosis of patients with AAV.
We conducted this study according to the Declaration of Helsinki and the Ethical Guidelines
for Epidemiological Research in Japan. Written informed consent was obtained from
each participant, and the study protocol was approved by the ethics committee at each
participating hospital (refer to Acknowledgements). This study was registered with
the University Hospital Medical Information Network Clinical Trials Registry (UMIN000001648).

Patients were evaluated at months 3, 6, 12, 18, and 24 and at relapse, and the following
data were collected: vital status, BVAS 2003, laboratory data, treatments, and adverse
events. The Vascular Damage Index score was recorded at months 6, 12, and 24. Chest
radiography, thoracic computed tomography, arterial blood gas analysis, and respiratory
function data were collected at months 12 and 24 in patients with pulmonary involvement.
Observation was completed in March 2013. Only the baseline data are included in this
study; the results from analyses of follow-up data will be reported separately.

The site investigators completed and sent the electronic case report form for each
patient to the RemIT-JAV data centre at the Department of Medicine and Clinical Science,
Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences,
Okayama, Japan.

EMEA classification algorithm for AAV

The enrolled patients were classified using the stepwise EMEA algorithm as described
previously [2,4]. Briefly, the ACR criteria and Lanham criteria for EGPA were applied first. Patients
who did not fulfil the criteria for EGPA were classified as having GPA if they met
the ACR criteria for GPA or the CHCC histological definition for GPA or if they showed
histology compatible with the CHCC definition for MPA or ANCA positivity with either
of the EMEA-defined GPA surrogate markers. The remaining patients were classified
as having MPA if they had clinical features and histology compatible with small-vessel
vasculitis without the GPA surrogate markers. In addition, ANCA-positive patients
who had the EMEA-defined surrogate markers for renal vasculitis were classified as
having RLV, a variant form of MPA. The rest of the patients without histology compatible
with the CHCC definition of classic polyarteritis nodosa or typical angiographic features
of classic polyarteritis nodosa were categorised as having unclassifiable vasculitis.

To identify a subset of unclassifiable vasculitis with AAV-related nongranulomatous
pulmonary lesions, we defined exploratory surrogate markers for alveolar haemorrhage
and ILD and then applied them to the EMEA-defined unclassifiable patient population.
Surrogate markers for these conditions were as follows: haemoptysis or alveolar haemorrhage
evaluated by bronchoscopic examination; or ILD diagnosed by chest X-ray or thoracic
computed tomography.

Statistical analysis

We used the baseline data of the patients enrolled in this study for statistical analysis.
The primary purpose of this analysis was to determine the demographic and clinical
characteristics of Japanese patients with AAV. Categorical variables were compared
using Fisher’s direct probability test, and continuous variables were compared using
Student t test or the Mann–Whitney U test depending on data distribution. P < 0.05 was considered significant for statistical analyses between two categories.
When comparing among three categories, statistical significance was determined by
P < 0.05/3 using Bonferroni correction to avoid multiplicity. All statistical analyses
were performed by a biostatistician using the Statistical Package of JMP for Windows
software (version 8.0.2; SAS Institute Inc., Cary, NC, USA).

Results

Classification of 156 Japanese patients with AAV according to the EMEA algorithm

In total, 159 patients with AAV were initially enrolled in the RemIT-JAV study. Three
patients were then excluded; two patients did not undergo treatment, and one patient
had been diagnosed as having AAV and experienced a relapse at the time of enrolment.
As a result, 156 patients with newly diagnosed AAV were enrolled in the study.

Using the EMEA algorithm, we identified 14 patients with EGPA, 33 patients with GPA,
78 patients with MPA/RLV, and 31 patients who were unclassifiable (Figure 1). The average ages of the patients with EGPA (male/female, 5/9), GPA (male/female,
12/21), and MPA/RLV (male/female, 35/43) were 58.0, 63.6, and 71.1 years, respectively
(Table 1). Patients with MPA/RLV were significantly older at the time of presentation than
those with EGPA and GPA (P < 0.017 for both), and there was a female predominance for all AAV diseases. MPO-ANCA
was detectable in 50.0% of patients with EGPA, in 54.6% of those with GPA, and in
97.4% of those with MPA/RLV. In contrast, PR3-ANCA was detectable in none of the patients
with EGPA, in 45.5% of those with GPA, and in 2.6% of those with MPA/RLV.

Table 1.Comparison of demographics and disease states among AAV diseases

Surrogate markers involved in the classification according to the EMEA algorithm

Of the patients classified with GPA, 22 patients fulfilled the ACR criteria, two patients
showed CHCC-defined GPA histology, three patients showed CHCC-defined MPA histology
in the presence of EMEA-defined GPA surrogate markers, and six patients were positive
for ANCA in the presence of GPA surrogate markers. Consequently, nine patients were
classified by the presence of GPA surrogate markers, mostly by the presence of chronic
sinusitis or otitis media (Table 2). Of the patients classified with MPA/RLV, 45 patients showed histology compatible
with small-vessel vasculitis without GPA surrogate markers and 33 patients were positive
for ANCA with EMEA-defined surrogate markers for renal vasculitis. Three patients
with allergic rhinitis, but not asthma, who had eosinophilia of peripheral blood and
tissue were considered to have unclassifiable vasculitis. The eosinophilic vasculitis
was confirmed histologically in all three patients, and two of these three patients
were MPO-ANCA-positive. The exploratory surrogate markers for AAV-related nongranulomatous
pulmonary lesions were positive in 16 of the 31 unclassifiable patients; three patients
had both markers (alveolar haemorrhage and ILD), and 13 patients had only ILD. These
16 patients were all positive for MPO-ANCA.

Table 2.Surrogate markers in nine patients who were classified with granulomatosis with polyangiitis
using these markers

Organ involvement of 156 Japanese patients with AAV

Patterns of organ involvement defined by the BVAS 2003 scoring system were described
and compared among patients with EGPA, GPA, and MPA/RLV (Table 3). Most patients with AAV presented with constitutional symptoms. Cutaneous and nervous
system manifestations were most common in patients with EGPA (71.4% and 92.9%, respectively).
Ear, nose, and throat manifestations and chest manifestations were characteristic
of patients with GPA (84.9% and 66.7%, respectively). It is noteworthy that ILD is
not included in the BVAS 2003 scoring system. Renal manifestations developed frequently
in patients with MPA/RLV (91.0%) but also in patients with GPA (63.6%). The mean serum
creatinine level and prevalence of ILD in patients with MPA/RLV was higher than that
in patients with EGPA and GPA, with a significant difference between MPA/RLV and EGPA
for both (Table 1). Unclassifiable patients had cutaneous (32.3%), renal (48.4%), and nervous system
(48.4%) manifestations (Table 3).

Differences in clinical features between MPO-ANCA-positive and PR3-ANCA-positive AAV

We compared the demographic and clinical features of Japanese patients with AAV who
had MPO-ANCA and those who had PR3-ANCA (Table 4). Patients with MPO-ANCA were significantly older at the time of presentation (P = 0.012) and had a higher rate of ILD (P = 0.0015). The mean serum creatinine level was numerically higher in patients with
MPO-ANCA. According to the BVAS 2003 scoring system, MPO-ANCA-positive patients had
more cutaneous (P = 0.046) and renal (P = 0.010) manifestations and fewer ear, nose, and throat manifestations (P < 0.0001) with statistical significance.

Table 4.Comparison of demographics and disease manifestations in MPO-ANCA-positive and PR3-ANCA-positive
patients

Of the 33 patients with GPA, 15 patients, 12 patients, and three patients were positive
for MPO-ANCA alone, for PR3-ANCA alone, or for both ANCAs, respectively, but three
patients were negative for ANCA. Patients with GPA who had MPO-ANCA had a numerically
higher rate of renal disease (86.7%) than those with PR3-ANCA (41.7%). The mean serum
creatinine level of patients with MPO-ANCA-positive GPA (2.05 ± 0.35 mg/dl) was also
numerically higher than that of patients with PR3-ANCA-positive GPA (1.03 ± 0.39 mg/dl).

Clinical features of patients with or without interstitial lung disease

We compared the demographic and clinical characteristics of the patients with and
without ILD (Table 5). MPO-ANCA was found significantly more frequently (P < 0.001) and PR3-ANCA was found less frequently (P = 0.038) in patients with ILD. These patients also tended to have more early systemic
diseases and less generalised or severe diseases (P = 0.059) and had significantly lower BVAS (P = 0.019). The mean serum creatinine level and rates of patients with constitutive
symptoms were similar between the two subgroups. The patients with ILD also had statistically
fewer ear, nose, and throat (P = 0.006) and cardiovascular (P = 0.012) manifestations.

Discussion

This is the first study to apply the EMEA algorithm to prospectively collected and
high-quality data of AAV patients outside Europe and to elucidate the clinical phenotypes
of the disease. In this study, 156 Japanese patients with newly diagnosed AAV were
enrolled from major universities and referring hospitals across Japan and classified
according to the EMEA algorithm. The results clearly indicated that MPO-ANCA-positive
MPA/RLV was the most common form of AAV in the Japanese population, and more than
one-half of the patients with EMEA algorithm-classified GPA showed MPO-ANCA positivity.
In addition, we showed that ILD was a common manifestation in Japanese patients with
AAV, especially in those with MPA.

The predominance of MPA/RLV and MPO-ANCA positivity in the Japanese population is
in marked contrast to the results of studies previously reported from European countries
and the United States [3,17-19]. Watts and colleagues validated the EMEA algorithm using 80 paper cases that were
originally written for evaluation of the BVAS system for systemic vasculitis with
some modifications, representing the relative frequency of AAV in their communities
as follows: GPA > MPA > EGPA [4]. It is therefore indispensable and important to evaluate the utility of the EMEA
algorithm in ethnicities outside Europe, as we did in this study. We found some difficulties
in the classification between GPA and MPA with the EMEA algorithm; for example, of
the nine patients classified as having GPA owing to the presence of GPA surrogate
markers, five had chronic sinusitis in which granulomatous inflammation was not proven
by histology. Because chronic sinusitis is a common disease and because fixed pulmonary
infiltrates and otitis media are sometimes observed in AAV diseases other than GPA,
classification of AAV using GPA surrogate markers should be cautiously applied in
the countries or regions where MPA is more prevalent than GPA.

Within the spectrum of AAV, there are interesting geographic differences in the relative
incidence of GPA versus MPA as well as of MPO-ANCA versus PR3-ANCA positivity [20]. In European countries, the incidence of GPA is approximately 4.9 to 10 per million,
depending on the geographic location, with higher incidences reported in more northern
countries and lower incidences in more southern countries [21,22]. A similar inverse relationship between GPA and MPA has been observed in the Southern
Hemisphere [22]. A higher incidence of MPA/RLV than GPA and the predominance of MPO-ANCA found in
the Japanese and Chinese AAV populations [3,5] could be related to the lower latitude of these countries.

GPA and MPA are heterogeneous entities with overlapping phenotypes. Recent studies
have indicated that the classification system based on ANCA specificity (that is,
MPO-ANCA versus PR3-ANCA) may better reflect the phenotypic spectrum of AAV. Cluster
analysis of patients with newly diagnosed GPA and MPA from five clinical trials showed
that the ANCA specificity classification may be more strongly associated with outcomes
such as death and relapse rate than the traditional GPA-MPA separation [23]. Moreover, compared with the CHCC definition and the EMEA algorithm, ANCA specificity
was more predictive of relapse in patients with biopsy-proven AAV; patients with PR3-ANCA
were almost twice as likely to experience a relapse as those with MPO-ANCA [24]. In this regard, it is intriguing that a genome-wide association study of a European
population revealed the presence of genetic distinctions between GPA and MPA that
are associated with ANCA specificity [25]. Because of the limited number of patients with PR3-ANCA in our RemIT-JAV cohort,
we were not able to perform cluster analysis within this database. We are currently
implementing another large-scale cohort study of Japanese patients with AAV, and the
combined database will enable us to clarify an association between ANCA positivity
and clinical characteristics of AAV in the Japanese population.

MPO-ANCA may contribute to the severity of chronic renal injury and the prevalence
of ILD in patients with AAV. Studies of renal biopsy specimens from patients with
AAV have demonstrated a higher prevalence and/or severity of renal lesions in MPO-ANCA-positive
patients compared with PR3-ANCA-positive patients [26]. These reports are in line with our findings that the mean serum creatinine level
of MPO-ANCA-positive patients was numerically higher than that of PR3-ANCA-positive
patients (1.94 versus 1.22 mg/dl).

A number of case reports and small case series have indicated that ILD developed more
frequently in patients with MPO-ANCA-positive AAV, mainly in those with a diagnosis
of MPA, compared with patients with PR3-ANCA-positive AAV [27,28]. A high ratio of MPO-ANCA positivity to PR3-ANCA positivity and a high prevalence
of ILD have been reported in Asian countries [8-10], and vice versa in northern European countries; ILD was reported in 7.2% of all patients with MPA
in the United Kingdom and in less than 10% in other European countries [11,12]. In this study, we confirmed a high prevalence of ILD in Japanese patients with AAV.
These patients were categorised as having a milder form (that is, more early systemic
and less generalised or severe diseases) and lower disease activity according to the
BVAS (Table 5), partially because ILD is not included in these definitions. Investigation of the
clinical courses and prognoses of patients with ILD will shed more light on the relevance
of ILD in the severity and activity of AAV.

We identified 16 unclassifiable AAV patients with ILD who were eligible for the EMEA
algorithm because they were MPO-ANCA-positive, had symptoms and signs compatible with
AAV such as general symptoms, and could not be diagnosed as having other diseases.
A previous study reported that MPO-ANCA seroconversion from negative to positive occurred
in 10% of patients with ILD in their clinical courses and that some patients with
MPO-ANCA and ILD eventually developed typical MPA [29]. On the other hand, vasculitis was proven in five of 15 biopsy specimens of MPO-ANCA-positive
patients with pulmonary fibrosis [13]. These data indicate that patients with unclassifiable AAV and ILD could be classified
as having MPA. Further investigation is required to pursue this possibility.

This study has some limitations. The number of patients evaluated was limited, and
the patient data were collected from the university and referral hospitals in large
cities in Japan, which might cause tertiary care biases for the relative frequency
of AAV diseases.

Conclusions

MPO-ANCA-positive MPA/RLV is the most common component of AAV in the Japanese population,
and more than one-half of patients with GPA are also positive for MPO-ANCA. ILD is
an important clinical manifestation in Japanese patients with AAV. Unclassifiable
vasculitis with MPO-ANCA positivity and ILD may represent a novel variant of MPA.
These data confirm the substantial difference in clinical and ANCA serological features
of AAV between western countries and Asian countries, including Japan, and indicate
that further investigation and discussion are required from a global perspective for
a better AAV classification system that can be applied to all geographic areas and
ethnicities.

Consent

This study was approved by the following ethical committees: Ethics Committee of the
Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences;
Medical Research Ethics Committee of Tokyo Medical and Dental University; Kyoto University
Ethics Committee Review Board; Ethics Committee of Kagawa University; Ethics Committee
of Juntendo University School of Medicine; Ethics Committee University of Tsukuba
Hospital; Ethics Committee of St. Marianna University School of Medicine; Kanazawa
University Ethical Committee; Ethics Committee of the University of Tokyo; Ethics
Committee of Kyorin University School of Medicine; Saitama Medical Center Hospital
Ethics Committee; Research Ethics Committee of the University of Miyazaki; Local Ethics
Committee of Toho University; Ethics Committee of Kobe University Hospital; Ethics
Committee of Kitano Hospital, The Tazuke Kofukai Medical Research Institute; Shimane
University Institutional Committee on Ethics; Ethics Review Committee of Nagoya City
University Graduate School of Medical Sciences; Ethics Committee of Ehime University
Graduate School of Medicine; Ethics Committee of Jichi Medical University; Ethics
Committee of Kyoto Prefectural University School of Medicine; Ethics Committee of
Tokyo Medical University Hachioji Medical Center; Ethics Committee of Kitasato University
Hospital; and Ethics Committee of Hamamatsu University School of Medicine.

Appendix

Research Committee of Intractable Vasculitis Syndrome of the Ministry of Health, Labour,
and Welfare of Japan: in addition to the authors, the following investigators and
institutions participated in this study: Department of Rheumatology and Clinical Immunology,
Saitama Medical Center, Saitama Medical University (Koichi Amano); Department of Nephrology,
Faculty of Medicine, University of Tsukuba (Kunihiro Yamagata); Department of Hemovascular
and Artificial Organs, Faculty of Medicine, University of Miyazaki (Shouichi Fujimoto);
Department of Respiratory Medicine, Toho University Omori Medical Center (Sakae Homma);
Department of Clinical Pathology and Immunology, Kobe University Graduate School of
Medicine (Shunichi Kumagai); Center for Nephrology and Urology, Division of Nephrology
and Dialysis, Kitano Hospital, Tazuke Kofukai Medical Research Institute (Eri Muso);
Department of Rheumatology, Shimane University Faculty of Medicine (Yohko Murakawa);
Division of Rheumatology, Department of Medical Oncology and Immunology, Nagoya City
University Graduate School of Medical Science (Shogo Banno); Department of Bioregulatory
Medicine, Ehime University Graduate School of Medicine (Hitoshi Hasegawa); Division
of Nephrology, Department of Internal Medicine, Jichi Medical University (Wako Yumura);
Department of Cardiovascular Medicine, Kyoto Prefectural University School of Medicine
(Hiroaki Matsubara); Division of Nephrology, Tokyo Medical University Hachioji Medical
Center (Masaharu Yoshida); Department of Dermatology, Kitasato University School of
Medicine (Kensei Katsuoka); and Third Department of Internal Medicine, Division of
Immunology and Rheumatology, Hamamatsu University School of Medicine, Hamamatsu (Noriyoshi
Ogawa).

Authors’ contributions

KS was responsible for conception and design, data collection and analysis, and manuscript
writing. MY, MH, and TF were responsible for conception and design, data collection
and analysis, and critical revision. HD, YT, SI, HY, TW, and JH were responsible for
data collection and interpretation, and critical revision. YA and HM were responsible
for conception and design, data collection and analysis, and critical revision. All
authors read and approved the final manuscript.

Acknowledgements

This work was supported by grants from Research on Rare and Intractable Diseases,
the Ministry of Health, Labour and Welfare, Japan (nannti-ippann-004).